Comparative transcriptome analyses reveal insights into catkin bloom patterns in pecan protogynous and protandrous cultivars

In perennial plants such as pecan, once reproductive maturity is attained, there are genetic switches that are regulated and required for flower development year after year. Pecan trees are heterodichogamous with both pistillate and staminate flowers produced on the same tree. Therefore, defining genes exclusively responsible for pistillate inflorescence and staminate inflorescence (catkin) initiation is challenging at best. To understand these genetic switches and their timing, this study analyzed catkin bloom and gene expression of lateral buds collected from a protogynous (Wichita) and a protandrous (Western) pecan cultivar in summer, autumn and spring. Our data showed that pistillate flowers in the current season on the same shoot negatively impacted catkin production on the protogynous ‘Wichita’ cultivar. Whereas fruit production the previous year on ‘Wichita’ had a positive effect on catkin production on the same shoot the following year. However, fruiting the previous year nor current year pistillate flower production had no significant effect on catkin production on ‘Western’ (protandrous cultivar) cultivar. The RNA-Seq results present more significant differences between the fruiting and non-fruiting shoots of the ‘Wichita’ cultivar compared to the ‘Western’ cultivar, revealing the genetic signals likely responsible for catkin production. Our data presented here, indicates the genes showing expression for the initiation of both types of flowers the season before bloom.


Introduction
Pecan (Carya illinoinensis) trees are monoecious, forming staminate and pistillate flowers on separate inflorescences. Pollination in pecan is anemophilous (wind pollinated) and trees manifest dichogamy, meaning that staminate and pistillate flowers of an individual genotype are of 2019, from each tree, 40 shoots were selected (20 shoots producing and 20 shoots with no pistillate flower at the time of the data collection). The number of individual catkins were counted on each shoot. The fruiting or non-fruiting status of every individual shoot for the previous year was also recorded by the presence of a dried rachis or rachis scar.

Bud microscopy
To see the morphological differences in formation of catkin primordia in bud tissues, 15 dormant buds from 'Western' and 15 dormant buds from 'Wichita' cultivars collected in February were excised. Samples were fixed in 2.5% glutaraldehyde solution buffered with 0.1M imidazole-HCl and stored at 4˚C. Vertical dissection of individual dormant buds was performed using a clean, stainless steel razor blade into two halves, samples were placed on sands under water and were first examined using a model M165FC stereofluorescence microscope (Leica Microsystems, Buffalo Grove, IL) using the GFP1 and Violet filter sets to evaluate favorable planes through the presumptive tissues. Then, for Laser scanning confocal microscopy, the cut surfaces of the selected buds were mounted on the coverslip surface of glass-bottom microwell dishes (MatTek Corp., Ashland, MA) and examined with a model TCS SP5II confocal microscope system (Leica Microsystems, Exton, PA) using the 488 nm Argon laser line for fluorescence excitation and a 20x long working distance objective lens. Fluorescence emission was collected in three channels, green from 500-570 nm, yellow-orange from 580-620 and red from 650-720 nm, as stacks of optical sections approximately 30-40 micrometers deep. Final images of comparable buds were displayed as graphic overlays of the three channels.

Plant material for RNA-seq
Bud tissue samples from two pecan cultivars, 'Wichita' and 'Western', were collected in June 13 June 21, 2017, and September 16, 2016, and March 5, and March 22, 2018. Samples were collected from three individual trees for each cultivar from the same location as samples collected for microscopy. On each tree, four fruiting shoots and four non-fruiting shoots were randomly selected for sample collection. The lateral buds were removed and frozen directly into liquid nitrogen. For each tree, bud samples from fruiting shoots were bulked together and samples from non-fruiting shoots of each tree were bulked together. Samples from different trees kept separated and used as replicates. In March, there was not yet any active growth or pistillate flowers formed, so the fruiting/non-fruiting samples were labeled based on the shoot fruiting status from the previous year (i.e., shoots were categorized into those that produced fruit (fruiting) and those that produced no fruit (non-fruiting) the previous season). Samples were homogenized separately with mortar and pestle using liquid nitrogen. Approximately 100 mg of frozen tissue was utilized for RNA extraction. Total RNA was extracted using plant/ fungi total RNA purification kit (Norgen Biotek, Ontario, Canada) according to manufacturer's instruction. All samples were DNase treated using the Qiagen DNAse kit to remove any DNA contamination. Sequencing libraries were constructed using an Illumina TruSeq stranded mRNA library kit (20020595) and TruSeq RNA UD Indexes (20022371) using

RNA-seq analyses
All reads were trimmed for adapter, quality, and length. Reads were trimmed using quality scores with 0.05 limit through automatic and/or trim adapter list to remove the read-through adapters. Then all the reads were trimmed to remove a maximum of 2 ambiguous bases at either 3'or 5'end of the reads and reads shorter than 5 nucleotides length were discarded. Trimmed reads were then mapped to the pecan reference genome of '87MX 3.2-11' V.1.1 (Phytozome). Read alignment performed with a mismatch cost of 2, insertion and deletion costs of 3, length and similarity fraction of 0.8 and a maximum number of 10 hits for a read. Differential gene expression analyses were performed using CLC Genomics workbench 12.0.2 (https://digitalinsights.qiagen.com). All transcriptomes were analyzed for a selection of 1229 genes due to their known function in flowering in other species (Arabidopsis thaliana and Juglans regia) in addition to highly expressed genes in pecan 'bud and catkin' and 'bud and pistillate' tissue specific analyses. All the analyses were based on a log2 fold change of higher than 1.5 and adjusted p-value of less than 0.05. The RNA-Seq data reads are available in NCBI Bio-Project PRJNA782058.

Staminate bloom patterns and statistical analyses
A Poisson regression analysis was carried out to study the catkin bloom patterns. The link function was the square root function. The regression model includes the fruiting and nonfruiting, and variety as independent variables, and the number of catkins as the response. In order to correct for the heteroscedasticity due to the shoots, an issue detected by the residual analysis, the model includes a variance component for each shoot. The numerical analysis was performed with Proc Glimmix, SAS 9.4, with the statement random _resid_ /group = shoot.

qRT-PCR
RNA was extracted from the same tissues using the method explained above. Extracted RNA were DNAse treated to remove any contaminant DNA. For each sample, 15 μl of RNA was mixed by 2 μl of 10X DNase Buffer, 1 μl of DNase I and 1μl of sterile water according to the manufacturer protocol (Deoxyribonuclease I, Amplification Grade, Invitrogen, Grand Island, NY). The reaction tube was incubated at room temperature for 15 min, then 1 μl of 25 mM EDTA solution was added to each reaction followed by the second incubation at 65˚C for 10 min. The DNase-treated RNA was quantified using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE) and was stored at -80˚C freezer. 20 ng of DNase-treated RNA was utilized for synthesizing the complementary DNA (cDNA) using a SuperScript1 IV Reverse Transcriptase kit (Invitrogen, Carlsbad, CA). For the initial reaction 1 μl of 50 μM oligo dT primer, and 1.0 μl of 10 mM dNTP mix was added to 20 ng DNAse-treated RNA. Sterile water was added to bring the volume to a total of 14 μl. Reaction was incubated at 65˚C for 5 min followed by a one-minute incubation on ice and vortexed for 3 seconds. Then, 4 μl of 5X SuperScript™ IV buffer, 1 μl of SuperScript™ IV Reverse Transcriptase and 1 μl of 100 mM DTT were added to the reaction tube based on the manufacturer protocol (Life Technologies, Grand Island, NY). The reaction tubes (total volume of 20 μl) were then incubated at 52.5˚C for 10 minutes followed by another 10-minute incubation at 80˚C. cDNA was stored at -20˚C for qPCR assays. Gene specific primers and probes were designed for GI, MS2, STM, KNAT6, TFL1 and CAL-A (Table 2) and synthesized by Integrated DNA Technologies (Coralville, IA) each with a unique fluorescent reporter dye. The ACTIN probe [14] was used as the housekeeping gene to normalize the qPCR analyses. Assays for each sample were performed in 3 technical and 3 biological replicates using iQ™ Multiplex Powermix (Bio-Rad). Quantitative PCR wells for each sample consisted of 20 ng (1 ul) of cDNA, 1X concentration of each probe, and sterile water into a 10 μl final volume. Pecan genomic DNA was used as a positive control, and at least three negative controls were included in each reaction plate. The qPCR was performed using a CFX96 touch real-time detection system (BioRad, Hercules, CA). The qPCR protocol was performed as explained by Thompson

Catkin counts and statistical analysis for 'Western' and 'Wichita'
To ascertain the impact of fruiting/non-fruiting on the production of catkin flowers the next year, we counted catkins from 'Western' and 'Wichita' trees. The previous year fruiting status, pistillate flower formation on the same year and the catkin counts were recorded and taken into consideration for catkin bloom analyses. The data from this study indicated that in 2019 there were an average of 5.0 individual catkins on 'Wichita' shoots that were non-fruiting in 2018 compared to the 8.2 individual catkins on 'Wichita' shoots that were fruiting in 2018 ( Fig 1A). However, there were an average of 5.0 individual catkins on 'Wichita' shoots that had pistillate flowers during spring 2019 and 9.5 individual catkins on 'Wichita' shoots with no pistillate flowers during spring 2019 ( Fig 1B). The number of catkins on a given 'Wichita' shoot was negatively correlated with the presence of pistillate flowers on the same shoot. In 'Wichita' the data indicates that there was a decrease in the number of catkins (p-value < 0.0001) when pistillate inflorescences were present (current year) on the same shoot. The presence of fruit the previous year also had a positive impact, and a significant increase of catkins was observed than compared to shoots that were not fruiting the year before (p-value < 0.01 for the previous  year). However, 'Western' catkin production was not significantly impacted by previous fruit development or current pistillate production on the shoots (Fig 1A and 1B, S1 Table).

Microscopy analysis confirms differences in anther development between protandrous and protogynous pecans
Catkins on protandrous cultivars such as 'Western', bloom and shed pollen generally earlier than protogynous cultivars such as 'Wichita'. The microscopy images of the buds collected in February 2019, before the beginning of the growing season, indicate the differences in catkin primordia formation/differentiation between 'Western' and 'Wichita' that occurred the previous season. In

RNA-seq analysis of the buds collected from pecan trees the season before bloom
Three genotyped 'Western' and three genotyped 'Wichita' pecan trees were used for collection of buds at different time points during the summer 2017 season. For each sample, four individual buds were collected and pulled. For the 'Fruiting' samples, buds were collected from shoots that were bearing fruit and for non-fruiting samples, buds were collected from shoots that were not bearing fruit. There was a total of 12 samples for each timepoint (three samples with four biological replicates/sample). For the 48 libraries represented in this study there were a total of 446,893,776 reads of which 96.4% were mapped to the 87Mx3-2.11 pecan genome [11]. All sequences have been deposited with NCBI BioProject PRJNA782058. June 2017, indicated a higher number of differentially expressed genes (DEGs) between the fruiting and non-fruiting samples from the 'Wichita' cultivar compared to the fruiting and non-fruiting samples from the 'Western' cultivar. As non-fruiting shoots of 'Wichita' most likely will produce more pistillate and catkin flowers the next year (compared to fruiting shoots) (Fig 1), some of the DEGs from the fruiting and non-fruiting 'Wichita' samples could be male specific genes. These data further emphasized the impact current pistillate production has on catkin production at the shoot level of the 'Wichita' cultivar in the same season while it had no significant effect on 'Western' (Fig 1).
In June 2017, there were only two DEGs between 'Western' non-fruiting and fruiting samples, MALE STERILITY 2 (MS2) and a non-characterized gene (S1A Fig). MS2, in some plant species, encodes a protein that is involved in male gametogenesis and is required for proper pollen development and in other plant species, is involved with male sterility [15,16]. pecan is yet to be studied, the expression pattern of MS2 in our study suggests the putative role of MS2 in pecan catkin production.
In comparison, during the month of June 2017, there were 36 differentially expressed genes between 'Wichita' (protogynous) non-fruiting and fruiting samples (S1 Fig). These genes, listed in Table 3, include genes that have been described in other species as necessary for initiation of both pistillate and staminate flowers [15][16][17][18][19][20]. From these 36 genes, 10 genes were upregulated in 'Wichita' non-fruiting sample and 26 were upregulated in 'Wichita' fruiting sample. Further investigation of the function of these genes in future studies, could reveal substantial understanding of flower initiation in pecan. In September, there were 10 unique DEGs between 'Western' non-fruiting and fruiting samples and 108 unique DEGs between 'Wichita' non-fruiting and fruiting samples.
S2 and S3 Figs indicate the log2 fold changes for the selected DEGs between 'Wichita' nonfruiting and fruiting samples in June 2017 and September of 2016. Many of these genes are known to specifically regulate flowering in other plant species. These genes included SUF4, EC1, UFO, MYB, MYB 24, and MYB108 [17][18][19][20][21][22]. In addition to genes that were anticipated to have role in male flower production, genes with known function in female flower development [23,24] were also differentially expressed in our samples collected in the season before bloom. ETR2 was upregulated in 'Western' non-fruiting buds collected in September 2016. Also, Table 3

. Selected DEGs between non-fruiting (NFr) and fruiting (Fr) samples collected in June 2017 and September 2016 from 'Wichita'(WI) and 'Western' (WE) cultivars ('Wichita' non-fruiting vs. 'Wichita' fruiting / 'Western' non-fruiting vs. 'Western' fruiting).
The samples in second column had the higher expression of the genes. The cut-off for the DEGs were set to a minimum log2fold change of 1.5 with adjusted p-value of less than 0.05. GRF7 and NFD4 were upregulated in 'Wichita' samples collected from non-fruiting and fruiting shoots respectively. There were several DEGs among our samples that may play a role in initiation of both male and female flowers such as AIL1, PRR9, etc., ( Table 3). The genes listed in this study are candidate genes for further investigation of their function in pecan flower initiation mechanisms. 'Western' vs 'Wichita'. Comparisons were also made between the 'Wichita' and 'Western' buds from June 2017 and September 2016. There were 107 DEGs between 'Wichita' and 'Western' non-fruiting samples. Forty-eight of these genes were specific and unique to the non-fruiting samples, and 16 DEGs were specifically upregulated in the 'Wichita' non-fruiting samples while the remainder were upregulated in 'Western' non-fruiting samples (S4 Fig)  (Table 4).

Collection time
There were 108 DEGs between 'Wichita' and 'Western' fruiting samples. Forty-nine of them were specific and unique to fruiting samples, 36 of these genes were upregulated in the 'Wichita' while the remainder were upregulated in 'Western' (Table 4). In September 2016, Table 4

. Selected DEGs between samples collected from 'Western' (WE) and 'Wichita' (WI) in June 2017 and September 2016 ('Wichita' fruiting (Fr) vs. 'Western' fruiting / 'Wichita' non-fruiting (NFr) vs. 'Western' non-fruiting).
Second column is the sample that had higher expression. The cut-off for the DEGs were set to a minimum log2fold change of 1.5 with adjusted p-value of less than 0.05. there were 127 DEGs between non-fruiting samples of 'Wichita and 'Western'. Forty-seven of these genes were specific to non-fruiting samples, out of which 29 genes were upregulated in the 'Wichita' non-fruiting samples and the remainder were upregulated in the 'Western' nonfruiting samples (18 genes) ( Table 4). In September 2016, there were 196 DEGs between 'Wichita' and 'Western' fruiting samples. 116 of these genes were specific to the fruiting samples among which 60 were upregulated in the 'Wichita' samples and the remainder of the 56 genes were upregulated in the 'Western' fruiting samples.

RNA-seq analyses of bud samples collected from 'Wichita' and 'Western' fruiting vs. non-fruiting during two sampling times in March.
In the season prior to flowering, several genes were differentially expressed between our samples that may have role in the initiation of male and/or female flowers. After the end of the growing season and the subsequent harvest, pecan trees are in a dormancy state during late fall and winter. Microscopy images from dormant buds collected in February 2019, showed that catkin primordia were fully developed in 'Western' buds as opposed to the 'Wichita' buds (Fig 2). To further investigate the flower initiation/formation genetic signals, additional bud samples were collected from the 'Western' and  (Table 5).
There were 71 DEGs between the 'Wichita' non-fruiting samples and the 'Western' nonfruiting samples (S7A Fig). Twenty of these genes were specific to non-fruiting samples. There were 100 DEGs between 'Wichita' fruiting samples and 'Western' fruiting samples. Forty-nine of these genes were specific to fruiting samples out of which 32 genes were upregulated in the 'Wichita' fruiting samples and 18 genes were upregulated in the 'Western' fruiting samples (S7B Fig). It is important to point out that in the early spring; catkin primordia are already completely formed in 'Western' buds while 'Wichita' buds continue to undergo the process of anther development. The function of these specific DEGs between samples collected from 'Wichita' and 'Western' in March 2018 require further evaluated for their roles in anther development.

Table 5. Selected DEGs between non-fruiting and fruiting sample (March 5 and March 22, 2018) from 'Wichita' (WI) and 'Western' (WE) cultivars.
Second column indicates the sample in which the given gene was significantly higher expressed compared to the other fruiting (Fr)/non-fruiting (NFr) status within the same genotype. The cut-off for the DEGs were set to a minimum log2fold change of 1.5 with adjusted p-value of less than 0.05.  The expression of canonical flowering genes such as CO, LFY, FD, FT, AP1, CAL-A, TFL1 and FLC were also detected in our samples (Fig 4).

Quantitative RT-PCR
To validate the accuracy and reproducibility of RNA-Seq analysis, six genes were randomly selected for qPCR analyses (Fig 5). Pearson correlation analyses was performed to find the correlation between the RNA-Seq normalized gene expression values (RPKM) and qPCR

Discussion
In perennial plants such as Carya illinoinensis, once reproductive maturity is attained, there are genetic switches that are regulated and required for flower development year after year. To understand these genetic switches and their timing in pecan, this study focused on developing buds' transcriptomes from two pecan cultivars, protandrous 'Western' and protogynous 'Wichita', the season before bloom and the season during bloom. 'Western' and 'Wichita' are widely grown in arid and semi-arid growing regions, such as the southwestern United States, where they are

PLOS ONE
included in most commercial orchards. The formation of both male and female flowers on the same pecan tree has made defining what genes are specifically responsible for pistillate and catkin (or both) formation challenging at best. In previous studies, it was found that pecan tree shoots that had previously produced fruit were less likely to produce pistillate flowers the following season as opposed to those branches (shoots) that did not produce fruit [7,8]. Our study showed that there were no statistical differences in catkin production on 'Western' trees as the shoots that had previously produced fruit had nearly the same number of catkins as the shoots that had not produced fruit (Fig 1). However, catkin production on 'Wichita' trees was profoundly different. There were statistically less catkins on 'Wichita' shoots that had pistillate flowers during the current season (Fig 1B). There were also statistically less catkins on the shoots that were not fruiting the previous season ( Fig 1A). The microscopy analysis (Fig 2) of buds from 'Western' and 'Wichita' trees showed the definitive difference in timing for anther development. Due to these differences in timing between these cultivars, we surmised that it may be possible to distinguish the genetic signals and the timing for flowering initiation in the buds during development. RNA-Seq analyses on the selected flowering genes from the bud samples collected from 'Wichita' and 'Western' cultivars revealed that the differences between the fruiting and nonfruiting shoots of 'Wichita' were more distinct than the fruiting and non-fruiting shoots of 'Western' in the season before bloom (June and September). Based on previous studies and microscopy images (Fig 2), we know catkin primordia formation begins in the dormant buds months in advance of bud break. However, the stage of catkin primordia development during the dormancy and budbreak periods differs between protogynous and protandrous genotypes. Therefore, it is reasonable to suggest that some of the flowering genes (RLP12, MYB108, ERF113, NFD4, WRKY75) that were differentially expressed between 'Wichita' and 'Western' samples (fruiting and non-fruiting) might have roles in catkin development (anther development) and could provide some preliminary clues for dichogamy in pecan. MYB genes have been shown to function in the formation of male flowers in other plant species. In other plant species, the MYB genes are involved in a transcriptional cascade that mediates stamen and pollen maturation [26][27][28][29]. The expression of these genes is mostly confined to sporophytic tissues of the stamen. The myb108 mutant exhibited reduced male fertility and this phenotype was associated with reduced pollen viability and delayed anther dehiscence [19]. Previous studies have shown that MYB24 and MYB108 have overlapping functions that facilitates stamen and pollen maturation in response to jasmonic acid (JA) [19]. In Arabidopsis, jasmonate is an essential key signal required for stamen and pollen maturation which leads to male fertility [30]. Previous studies in Arabidopsis suggested that MYB21 and MYB24 specifically regulate male fertility and the MYB21/24 double mutant exhibited male sterility due to defects in anther dehiscence, pollen maturation and filament elongation [26]. MYB21 and MYB24 along with another member of MYB family, MYB57, are GA-dependent stamen-enriched genes and the triple mutants in Arabidopsis conferred short stamen and lead to male sterility [27]. Other studies have shown that MYB21 and MYB24 promote petal and stamen development and have a role in gynoecium growth [28]. The auxin-induced protein 15A-like belongs to the ARG7 gene family. The ARG7 gene family can regulate plant growth hormones and affect pistil development [31]. Previous studies on pecan pistillate flower transcriptome also showed that ARG7 played a role in pecan floral organ development [9].
The catkin bloom patterns revealed that pistillate formation on 'Wichita' cultivar shoots negatively impacted catkin production on the same shoot in the same year. As 'Western' does not follow the same pattern, it is likely that the differentially expressed genes that were upregulated in the 'Western' samples are most likely related to catkin initiation/formation. Some DEGs with higher expression in the 'Western' fruiting shoots collected in June were most likely related to catkin initiation and genes highly expressed in the 'Western' fruiting samples from September could be putative genes for anther development. As it is expected for 'Wichita' catkin primordia to fully develop right before bloom, it is compelling that some higher expressed genes (MYB108, DCN1, RLP12, COL11, BHLH137, MIK2, RFK1, SRS1) in 'Wichita' samples compared to 'Western' samples in March were putative candidate genes for anther and pollen development in pecan. Comparison of 'Wichita' fruiting and non-fruiting samples also suggested SPL7 and MS2 as two other possible catkin specific genes. The SPL7 gene (SQUAMOSA PROMOTER BINDING-LIKE7) is one of the miR156-targeted SPL genes that is involved in the control of phase transition and flowering by regulating AP1 and FUL in Arabidopsis [32][33][34]. miR156 and its target, the SPL gene, control the aging pathway of flowering [35].
The aging pathway ensures flowering occurs, even under noninductive conditions [32,[36][37][38]. Overexpression of SPL7 in switchgrass promoted flowering whereas downregulation of this gene moderately delayed flowering [39]. The MALE STERILITY 2 (MS2) gene encodes a protein that is involved in male gametogenesis and is required for proper pollen development [15].
Shoots that did not bear fruit in 2017 for both 'Wichita' and 'Western' were most likely to bear pistillate flowers in May 2018. Our data showed a considerable number of DEGs between non-fruiting shoots of 'Wichita' and 'Western' in March 2018. Higher expression of some flower repressor genes (MYC2, SLN1) and BHLH137 (male sterility) in 'Wichita' non-fruiting samples were observed on March 22, 2018. Higher expression of these genes could potentially repress catkin development and bloom in 'Wichita' trees. Unfortunately, due to the destructive nature of sample collection for RNA-Seq, we lack the information regarding the ultimate fate of these buds.

Conclusions
To the best of our knowledge, no significant differences in the number of catkins between 'Western' and 'Wichita' have previously been reported. Here we show that there is a negative impact on staminate bloom on shoots that currently have pistillate flowers, suggesting that pistillate flower production in 'Wichita' may occur at the expense of catkin production but no differences were observed for 'Western' catkins. This study needs to be performed on additional protandrous and protogynous pecan cultivars to determine if there is indeed a direct relationship between pistillate flower and catkin production on protogynous pecan cultivars. Based on the previous studies, it was hypothesized that in pecan, flower initiation for both staminate and pistillate flowers occur one season before bloom. Our data presented gene expression evidence for the initiation of both flower types in the year before bloom, however, since both 'Wichita' and 'Western' non-fruiting shoots were expected to bear fruit the following year, and the caveat of alternate bearing is proportional (flowering shoots are less likely to produce fruit the following year), defining the pistillate specific genes were especially challenging. The current study specifically looked at the differentially expressed genes. Our previous analyses presented the expression of hundreds of known flowering genes, among which, several were known to be female specific genes in other species [10]. Further studies with more time points of sample collection are required for more comprehensive insight into the timing of pecan pistillate specific genes expression and a better understanding of the function of each gene in pecan.
Our data indicates that pecan flowering is evidently programmed to follow several precise 'clocks' (Fig 6) and the elements of these clocks will be used as genetic resources and background for better understanding of the flower initiation mechanisms in pecan. Further comprehensive studies of the selected candidate genes may enable researchers to understand the timing of floral initiation and ultimately assist in mitigation of alternate bearing effects and help the breeders to control or select specific traits of flowering in pecan.
Both protandrous and protogynous cultivars are dormant during the winter months. In spring, signals responsible for the production of female flowers occurs for both protogynous and protandrous trees. Protogynous trees begin anther development in their buds in early spring. Protandrous trees already have fully developed anthers in early spring. In late April and early May both protogynous and protandrous pecan trees have pistillate flower bloom and pollination and fertilization occur. After successful pollination, fruits develop throughout the summer. In a separate internal clock, flower signals are active in the bud for the next year and catkin primordia are forming in the bud. At the end of the summer, protandrous cultivars go through the development of anther primordia in the bud for the next year while no further development occurs in protogynous cultivars. Trees go dormant in winter and this cycle continues.